Proteins and Products

1
Chapter 4

• Proteins and Products

2
Search for new and novel proteins

• Going to extremes
• Tropical rain forests
• Yellowstone hot springs and soils
• Branches and pine needles in hot springs are a
  potential source of thermophilic lignin-degrading
  microbes and their enzymes
• These enzymes have commercial value in pulp and
  paper industry

3
Classes of Proteins

• Enzymes
• Depolymerization
• Enzymes that degrade large complex molecules such
  as lignin, starch and cellulose to smaller
  subunit compounds
• Amylase acts on starch to produce glucose
• What is the degradation product of cellulose?
• Lipases
• Proteases
• Hormones (chemical messengers)
• insulin

4
Examples of proteins used in other commercial
applications

• Textile industry
• Enzymes have replaced toxic chemical
• stone-washed denim
• Home products industry
• Enzymes added to Drano and Liquid Plumber
  replaces caustic chemicals to dissolve hair
  fibers, grease, etc. that plug drains

5
Adhesives

• Mussel adhesive protein
• Underwater glue
• Industries like 3M are trying to design and
  synthesize proteins in E. coli bacterium that
  mimic the natural proteins excreted by mussels

6
Protein structures

• Proteins are linear chains of amino acids linked
  together by a peptide bond
• Primary structure refers to the sequence of the
  different amino acids in the chain

-aa1- aa2- aa3- aa4-
H
aa1 -C-O-N-aa2-
O
7
Protein structure

• Secondary structure refers to the shape of the
  amino acid chain
• Beta sheets (ribbon)
• Alpha helix (cork screw)
• Tertiary structure refers to the shape the chain
  takes as a result of attractions between alpha
  helices and beta sheets in different sections of
  the chain

8
Protein structure
Chain 1

• Quaternary structure is a protein that contains
  more than one amino acid chain
• hemoglobin

Chain 2
9
Protein folding

• Folding of the peptide chain(s) confers structure
  to the protein
• Certain amino acids (aa) placed at specific
  intervals along the chain established how chain
  folds
• alpha helix
• beta sheet
• proline aa inserts a kink in the chain

10
Protein folding

• Structure controls protein function
• tangled plaques of Alzheimers disease may be
  the result in errors in protein folding
• Some proteins have sugar residues attached to the
  peptide chain
• glycosylation
• affects activity of protein
• found in eucaryotic cells

glycosylation
11
Diseases related to incorrectly folded proteins

• Cystic fibrosis
• mad cow disease (bovine spongiform encephalitis)
• A substitution of one amino acid for another
  along the peptide chain can affect how the
  protein folds.

12
Bioprocessing

• There are numerous steps between collecting raw
  material from nature and producing a commercial
  product

13
Large-scale cultivation of microorganisms

• Fermentation vessels or bioreactors has impeller
  that stirs the culture so bacteria in suspension
  get good access to dissolved nutrients and oxygen
  for growth.

14
Food processing proteins
15
Proteins in medical applications

• Insulin
• Recombinant insulin produced in E. coli avoids
  adverse reactions by our immune system in
  response to foreign substances (insulin recovered
  from pigs and cattle).
• Pharmaceutical industry has invested in this area
  because they can charge so much for use in
  treatments

16
Recombinant proteins produced by the
pharmaceutical industry
Application
Protein

• Erythropoietins
• Interleukin 1,2,3,4
• Human growth factor
• Tissue plasminogen factor
• vaccines

• Treatment of anemia
• Cancer, AIDS
• Treatment of growth deficiency in children
• Heart attack and stroke treatment
• Hepatitis B, herpes, malaria

17
Recombinant proteins produced in eucaryotic fungi
Fungi are good hosts for producing glycosylated
proteins since, as eucaryotic cells, they have
the molecular machinery to add sugars molecules
to peptide chains, whereas, bacteria do not have
this machinery
18
Protein engineering

• Shotgun approach
• induce mutations randomly into genes and then
  select organisms (bacteria) with protein products
  (enzyme) that, when assayed using an enzyme
  assay, gives the highest activity.
• Led to recovery of an enzyme that functioned in
  presence of high cyanide concentration.

19
Protein engineering

• Directed molecular evolution
• Induce mutation in a specific gene and select
  best protein from mutant gene for our application
  irrespective of its value to organism. (example
  insulin production in E. coli)

20
Protein engineering

• Hybrid proteins
• a piece of one protein linked to a piece of
  another protein in the same amino acid chain.
• This is often done to encourage E. coli cells to
  secrete protein into surrounding medium instead
  of holding it inside cell.
• This makes it easier to recover and purify the
  protein.
• An enzyme can be added after purification to cut
  off the part of the hybrid that signaled
  exportation.

signal sequence (only used to direct protein for
export out of cell
export
functional protein
protease cleavage
21
Upstream processing
Protein expression (transcription translation)
Protein export
22
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
dialysis
ion exchange
chromatography
23
Downstream processing

• Step 1-Recovery of protein from cells

Cell pellet
24
Cell lysis(when protein is still inside cell)
High-energy sound waves, High salt concentration
25
Downstream processing

• Step 2-Separation of protein from other culture
  medium components (purification)
• sometimes this is very time-consuming and costly
• purification is necessary because other medium
  components may interfere with protein function
  or cause adverse reaction if protein is
  introduced into a human

26
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
dialysis
ion exchange
chromatography
27
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
dialysis
ion exchange
chromatography
28
Dialysis
29
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
chromatography
dialysis
30
Chromatography
Separates protein of interest from other proteins
and medium components
31
Chromatography
Size-exclusion chromatography
Column filled with beads
detector
32
Chromatography
Ion-exchange chromatography
33
Identification of proteins separated in different
fractions
wells

• SDS-PAGE
• Sodium Dodecyl Sulfate Polyacrylamide Gel
  Electrophoresis

Works similar to agarose gel electrophoresis to
separate DNA and RNA fragments Used to following
process of separating protein of interest from
all other proteins
Demonstrates that you have a purified protein
34
Post-purification analysis

• Protein sequencing

Go to Quick-time movie
35
Post-purification analysis

• Protein sequencing

Go to Quick-time movie
36
Post-purification analysis

• Protein sequencing

Go to Quick-time movie
37
Post-purification analysis

• Protein sequencing

Go to Quick-time movie
38
Post-purification analysis

• Protein sequencing

unknown amino acid
Go to Quick-time movie
39
Post-purification analysis

• X-ray crystallography

40
Proteomics

• Proteomics refers to the evaluation of the
  complement of proteins expressed by a cell under
  a certain set of conditions
• Proteomes are compared under healthy and diseased
  states
• Variations in protein expression are correlated
  to onset or progression of a specific disease
• Proteomics applies electrophoresis, mass
  spectrometry and amino acid sequencing

41
Proteomics
Protein chips
Used to detect presence of specific proteins
10,000 probes
42
Pathway Tools overview diagram
43
(No Transcript)